Olumayokun A. Olajide

Acute respiratory infections (ARIs) are among the most common infectious diseases worldwide and represent a substantial clinical and socioeconomic burden. Despite their predominantly viral aetiology, inappropriate antibiotic prescribing remains common, highlighting the need for effective non-antibiotic treatment strategies. In this narrative review, we summarize the pharmacological properties and potential clinical relevance of Plantago lanceolata and Sisymbrium officinale in the symptomatic management of uncomplicated upper airway infections. A narrative literature search of PubMed/MEDLINE, Google Scholar, and the herbal monographs and assessment reports of the European Medicines Agency was conducted, focusing on experimental, clinical, and real-world evidence related to symptom relief, anti-inflammatory effects, and cough modulation. Emphasis was placed on recent in vitro studies investigating epithelial inflammatory responses and cough-related signalling pathways. In cell-based experimental models, both extracts reduced key inflammatory mediators, including prostaglandin E₂, tumour necrosis factor-α, and interleukin-6, in human tonsil epithelial cells stimulated with bacterial cell wall components. In addition, both plant extracts attenuated bradykinin-induced intracellular calcium signalling in bronchial epithelial cells, which is consistent with, but does not demonstrate, modulation of cough-related pathways. These in vitro findings provide a biological rationale for their traditional use in relieving sore throat, irritation, and cough, but they are not direct evidence of clinical efficacy, and clinical trials of these two extracts are lacking. Given the central role of symptom burden in driving healthcare utilization and antibiotic-seeking behaviour, effective symptomatic therapies are important. Whether herbal medicinal products with anti-inflammatory and cough-modulating properties improve symptom control and thereby support antimicrobial stewardship remains to be demonstrated. Future research should include randomized controlled trials with validated symptom outcomes and real-world studies evaluating the impact of these extracts on antibiotic prescribing in clinical practice.

Topical non-steroidal anti-inflammatory drugs (NSAIDs) such as flurbiprofen and benzydamine may be used for the symptomatic relief of pharyngitis, triggered by infections. The onset and spectrum of anti-inflammatory activity of both drugs were compared in human tonsil (HTEpiC) and bronchial epithelial (BEAS-2B) cells stimulated with poly I:C against key inflammatory mediators. Cells were stimulated with poly I:C for 24 h and treated with flurbiprofen or benzydamine for 20 s, 2 min and 5 min. Production of prostaglandin E2 (PGE₂), tumour necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), interleukin-1 beta (IL-1β), interleukin-8 (IL-8), interleukin-18 (IL-18), monocyte chemotactic protein-3 (MCP-3) and C-X-C motif chemokine ligand 10 (CXCL10), as well as cyclooxygenase-2 (COX-2) protein expression, was evaluated. Flurbiprofen significantly (p < 0.05) reduced PGE₂ production and COX-2 expression in both cell types within 20 s of treatment and maintained these effects at 2 and 5 min. However, benzydamine produced delayed, inconsistent inhibition. Although both drugs reduced poly I:C induced IL-6, IL-1β, IL-8 and IL-18 production, the magnitude and onset were greater with flurbiprofen. Flurbiprofen also reduced MCP-3 and CXCL10 in both cell types, alongside significant inhibition of caspase-1 activity. Benzydamine showed limited or no effect and less inhibition of caspase-1. With rapid suppression of the COX-2/PGE₂ pathway, rapid onset and a broad inhibition spectrum, flurbiprofen more comprehensively modulates inflammatory processes in epithelial respiratory models.

Symptoms of respiratory tract infections (RTI) caused by viral or bacterial triggers include sore throat and cough. Plantago lanceolata (ribwort plantain) is used in herbal medicine to treat symptoms of RTI. This study evaluated the pharmacological actions of P. lanceolata extract (PL) and StrepCough Plantain-Dry cough syrup (PLS) in in vitro models of sore throat and cough. Human tonsil epithelial cells (HTEpiC) cells were stimulated with a combination of lipoteichoic acid and peptidoglycan in the presence or absence of PL or PLS. Levels of TNF-α, IL-6, PGE₂, and COX-2 were measured using ELISA, while phospho-p65 protein levels were measured using Lumit^® immunoassay. The effects of PL and PLS on bradykinin-induced inflammation and increased intracellular Ca²⁺ were investigated in bronchial epithelial cells (BEAS-2B), cells over-expressing TRPV1. Results showed that the pre-treatment of HTEpiC cells with PL and PLS resulted in a significant (p < 0.05) reduction in the LTA + PGN-induced increased production of TNFα, IL-6, PGE₂, as well as COX-2 and phospho-p65 protein levels. Bradykinin-induced increased levels of TNFα, IL-6, and PGE₂ in BEAS-2B cells over-expressing TRPV1 were also reduced by PL and PLS, while reducing levels of intracellular Ca²⁺. This study has demonstrated that extract of P. lanceolata and StrepCough Plantain-Dry cough syrup reduce inflammation in human tonsil epithelial cells. The extract and syrup also reduced TRPV1-mediated inflammation and increased intracellular Ca²⁺ in BEAS-2B bronchial epithelial cells. This study has provided pharmacological evidence for the use of P. lanceolata in healthcare interventions to treat symptoms of respiratory tract infection.

Background: Acute respiratory distress syndrome (ARDS) is characterised by inflammation with the accompanying release of pro-inflammatory cytokines. AC186 is an oestrogen receptor agonist, which has shown anti-inflammatory activity. This study investigated effects of AC186 on poly I:C-induced inflammation in human bronchial epithelial (BEAS-2B) cells.Methods: Supernatants from poly I:C-stimulated BEAS-2B cells treated with AC186 (1.25, 2.5 and 5 µM) were analysed for levels of tumour necrosis factor-alpha (TNFα), interleukins-6 (IL-6), -1β (IL-1β) and −8 (IL-8), using ELISA. Protein expression of phospho-p65 NF-κB was evaluated using Lumit^® Immunoassay, while nuclear localisation, DNA binding and transcriptional activity of NF-κB were evaluated using immunofluorescence, transcription factor ELISA and reporter gene assays, respectively. In cell ELISAS were used to determine effects on NLRP3 and caspase-1 proteins while Caspase-Glo^® 1 inflammasome assay was used to determine whether AC186 influenced caspase-1 activity. Experiments were conducted to evaluate effects on ATP production and caspase 3/7 activity.Results: AC186 produced significant (p < 0.05) reduction in elevated levels of TNFα, IL-6, IL-1β and IL-8 in BEAS-2B cells, in comparison with poly I:C stimulation alone. Increased phosphorylation of p65 was significantly (p < 0.01) reduced in the presence of AC186 (2.5 and 5 µM), while nuclear localisation of p65, as well as DNA binding and transactivation were blocked with 2.5 and 5 µM of the compound. AC186 (2.5 and 5 µM) reduced protein levels of both NLRP3 inflammasome and caspase-1, as well as caspase-1 activity. Co-administration of ICI 182780 (10 nM) with AC186 (5 μM) prior to stimulation with poly I:C resulted in higher levels of TNFα and IL-6 secretion, compared to AC186 pre-treatment alone. Following incubation of AC186 (2.5 and 5 μM) with poly I:C-stimulated BEAS-2B cells for 72 h, there was significant improvement in viability as well as reduction in caspase 3/7 activity, in comparison with poly I:C alone.Conclusion: These results suggest that AC-186 produced anti-inflammatory activity in poly I:C-stimulated BEAS-2 cells, through mechanisms involving inhibition of NF-κB and NLRP3/caspase-1/IL-1β activation. AC186 also protected BEAS-2B cells against poly I:C-mediated apoptosis and death suggesting that this compound have potentials in reducing inflammatory events associated with ARDS caused by viral infections.

The controversies surrounding animal experimentation, its application in pharmacological research has contributed immensely to drug discovery. The fact that most animal models share similar molecular targets with humans makes them essential regulatory requirements for pre-clinical pharmacology experiments for new drugs. Consequently, millions of experimental animals have been used globally. However, with the introduction of the replacement, reduction and refinement (3Rs) ethical framework to reduce experimental animal use and their suffering, investigators are required to adhere to their principles. Although in vitro models have been proposed as alternatives to animal models, there are limitations to their use, especially those related to pharmacokinetics under culture conditions. The use of stem cells, including induced pluripotent stem cells (iPSCs) reprogrammed from adult somatic cells, have been proposed as viable and reliable alternatives to animal experimentation and in vitro modelling because they can be reprogrammed into various cells for application in pharmacological research. In this respect, human induced pluripotent stem cells (hiPSCs) can be generated from patients with specific diseases, allowing the creation of in vitro disease models that mimic the cellular and molecular characteristics of the relevant disease. hiPSC-derived cells can also be employed in screening new drug candidates for efficacy and toxicity, thus allowing for personalised drug screening, and identifying drugs that are most likely to be effective and safe for a specific patient. Furthermore, hiPSCs have potential applications in identifying and validating potential drug targets by studying the cellular and molecular mechanisms underlying disease. However, despite promising applications of hiPSCs, there are challenges in ensuring the quality, safety, and reproducibility of hiPSC-derived cells, as well as in accurately modelling complex diseases and predicting long-term drug effects. Also, there are still ethical considerations related to the source of somatic cells used for re-programming. The lecture will throw more light on successful applications of hiPSCs in pharmacological research, as well as challenges with the use of iPSCs as alternatives to animal and in vitro experiments.

Background: AC-186 (4-[4-4-Difluoro-1-(2-fluorophenyl) cyclohexyl] phenol) is a neuroprotective non-steroidal selective oestrogen receptor modulator. This study investigated whether inhibition of neuroinflammation contributed to neuroprotective activity of this compound. Methods: BV-2 microglia were treated with AC-186 (0.65–5 μM) prior to stimulation with LPS (100 ng/mL). Levels of pro-inflammatory mediators and proteins were then evaluated. Results: Treatment of LPS-activated BV-2 microglia with AC-186 resulted in significant (p < 0.05) reduction in TNFα, IL-6, NO, PGE₂, iNOS and COX-2. Further investigations showed that AC-186 decreased LPS-induced elevated levels of phospho-p65, phospho-IκBα and acetyl-p65 proteins, while blocking DNA binding and luciferase activity of NF-κB. AC-186 induced significant (p < 0.05) increase in protein expression of ERβ, while enhancing ERE luciferase activity in BV-2 cells. Effects of the compound on oestrogen signalling in the microglia was confirmed in knockdown experiments which revealed a loss of anti-inflammatory activity following transfection with ERβ siRNA. In vitro neuroprotective activity of AC-186 was demonstrated by inhibition of activated microglia-mediated damage to HT-22 neurons. Conclusions: This study established that AC-186 produces NF-κB-mediated anti-inflammatory activity, which is proposed as a contributory mechanism involved in its neuroprotective actions. It is suggested that the anti-inflammatory activity of this compound is linked to its agonist effect on ERβ.

Nimbolide, a limonoid compound found in the neem plant, was investigated for effects on neuroinflammation in BV-2 microglia activated with lipopolysaccharide (LPS). Cultured BV-2 cells were treated with nimbolide (125, 250 and 500 nM) followed by stimulation with LPS (100 ng/ml). Results showed that nimbolide caused a significant reduction in the levels of TNFα, IL-6, IFNγ, NO/iNOS and PGE₂/COX-2 in LPS-activated BV-2 cells. Further experiments revealed that LPS-induced increased expression of phospho-p65 and phospho-IκBα proteins were reduced in the presence of nimbolide. Also, LPS-induced NF-κB acetylation, increased binding to consensus sites and transactivation, as well as phosphorylation of p38 and JNK MAPKs were reduced by nimbolide. Reduction of cellular ROS generation by nimbolide was accompanied by a reduction in gp91phox protein levels, while antioxidant effects were also observed through elevation in protein levels of HO-1 and NQO-1. It was observed that treatment of BV-2 microglia with nimbolide resulted in reduced levels of cytoplasmic Nrf2, which was accompanied by increased levels in the nucleus. Furthermore, treatment with this compound resulted in increased binding of Nrf2 to antioxidant responsive element (ARE) consensus sites accompanied by enhanced ARE luciferase activity. Knockdown experiments revealed a loss of anti-inflammatory activity by nimbolide in cells transfected with Nrf2 siRNA. Treatment with nimbolide resulted in nuclear accumulation of SIRT-1, while siRNA knockdown of SIRT-1 resulted in the reversal of anti-inflammatory activity of nimbolide. It is proposed that nimbolide reduces neuroinflammation in BV-2 microglia through mechanisms resulting in dual inhibition of NF-κB and MAPK pathways. It is also proposed that activation of Nrf2 antioxidant mechanisms may be contributing to its anti-inflammatory activity. Graphical Abstract

Skimmianine is a furoquinoline alkaloid which is found in the Zanthoxylum genus and also in other plants of the Rutaceae family. This study evaluated the effects of skimmianine on the production of pro-inflammatory mediators in LPS-activated BV-2 microglia. Cultured BV-2 cells were treated with skimmianine (10, 20 and 30 μM), followed by stimulation with LPS (100 ng/mL). Levels of TNFα and IL-6 in cell supernatants were measured using ELISA, while NO and PGE₂ levels were evaluated with Griess assay and EIA, respectively. Western blotting was used to determine the protein expression of iNOS, COX-2, phospho-p65 and phospho-IκBα. Results showed that Skimmianine reduced LPS-induced elevated the secretion of TNFα, IL-6, NO, and PGE₂, as well as the increased protein expression of iNOS and COX-2. Experiments to elucidate the mechanisms of the anti-neuroinflammatory activity of skimmianine revealed the significant inhibition of LPS-induced increased NF-κB-mediated luciferase activity. Pre-treatment with skimmianine also reduced LPS-induced the increased phosphorylation of NF-κB/p65 and IκBα proteins. Furthermore, skimmianine interfered with the binding capacity of NF-κB to consensus sites. Skimmianine pre-treatment protected HT-22 cells from toxicity induced by microglia-conditioned media, as well as increasing MAP-2 expression. The results of this study suggest that skimmianine inhibits neuroinflammation in LPS-activated microglia by targeting the NF-κB activation pathway. Skimmianine also produced neuroprotection against neurotoxicity induced by microglia-conditioned media.

Nimbolide, a limonoid compound found in the neem plant, was investigated for effects on neuroinflammation in BV2 microglia activated with LPS. Cultured BV2 cells were stimulated with LPS (100 ng/ml) followed by treatment with nimbolide (125, 250 and 500 nM). Results showed that nimbolide caused significant reduction in the levels of TNFα, IL-6, IFNγ, NO/iNOS, PGE₂/COX-2 in LPS-activated BV2 cells. Further experiments revealed that LPS-induced increased expression of phospho-p65 and phospho-IκBα proteins were reduced in the presence of nimbolide. Also, LPS-induced NF-κB acetylation, increased binding to consensus sites, and transactivation, as well as phosphorylation of p38 and JNK MAPKs were reduced by nimbolide. Reduction of cellular ROS generation by nimbolide was accompanied by reduction in gp91phox protein levels, while antioxidant effects were observed through elevation in protein levels of HO-1 and NQO-1. It was observed that treatment of BV2 microglia with nimbolide resulted in reduced levels of cytoplasmic Nrf2 which was accompanied by increased levels in the nucleus. Furthermore, treatment with this compound resulted in increased binding of Nrf2 to ARE consensus sites accompanied by enhanced ARE luciferase activity. Knockdown experiments revealed a loss of anti-inflammatory activity by nimbolide in cells transfected with Nrf2 siRNA. Treatment with nimbolide resulted in nuclear accumulation of SIRT-1, while siRNA knockdown of SIRT-1 resulted in reversal of anti-inflammatory activity of nimbolide. It is proposed that nimbolide reduces neuroinflammation in BV2 microglia through mechanisms resulting in dual inhibition of NF-κB and MAPK pathways. It is also proposed that activation of Nrf2 antioxidant as well as SIRT-1 deacetylation mechanisms may be contributing to its anti-inflammatory activity.